Homologous recombination between mammalian cellular sequences and their counterparts introduced by DNA transfer methods could play an important role in human gene therapy. It can also be used for gene-knockout which will provide a new genetic tool for analysis of gene function. Homologous recombination between input plasmid sequences and between input plasmid and chromosomal sequences has been demonstrated. In order for these methods to be of general utility it is necessary to enhance the frequencies of homologous recombination. This proposal is aimed at developing methods to achieve gene replacement and insertional inactivation (gene knockout) of mammalian genes by homologous recombination. Autonomously replicating vectors will be used to achieve extremely high frequencies of homologous recombination. A method to achieve hit-and-run recombination to modify cellular genes is proposed. Use of replicating and non-replicating vectors to achieve gene knockout are also proposed. A particular problem with mammalian cells is that not one but two copies of a gene have to be inactivated to observe a recessive phenotype. Use of double knock-out vectors and gene fusions are proposed to achieve this goal. Examination of repair of heteroduplexes, which are generated during the recombination process, in normal human cells and cells from patients who exhibit chromosomal fragility syndromes is proposed. Repair of single base mismatches, clustered base mismatches, single strand gaps and loops will be examined by using heteroduplexes prepared from autonomously replicating plasmids. Finally, a series of experiments aimed at developing a cell-free system to study repair of heteroduplexes is also proposed. Many of the experiments are based on previous successes in the applicant's laboratory.